Science-based evidence on pathways and effects of human exposure to micro- and nanoplastics

Abstract Human exposure to plastic particles has raised great concern among all relevant stakeholders involved in the protection of human health due to the contamination of the food chain, surface waters, and even drinking water as well as due to their persistence and bioaccumulation. Now more than ever, it is critical that we understand the biological fate of plastics and their interaction with different biological systems. Because of the ubiquity of plastic materials in the environment and their toxic potential, it is imperative to gain reliable, regulatory-relevant, science-based data on the effects of plastic micro- and nanoparticles (PMNPs) on human health in order to implement reliable risk assessment and management strategies in the circular economy of plastics. This review presents current knowledge of human-relevant PMNP exposure doses, pathways, and toxic effects. It addresses difficulties in properly assessing plastic exposure and current knowledge gaps and proposes steps that can be taken to underpin health risk perception, assessment, and mitigation through rigorous science-based evidence. Based on the existing scientific data on PMNP adverse health effects, this review brings recommendations on the development of PMNP-specific adverse outcome pathways (AOPs) following the AOP Users’ Handbook of the Organisation for Economic Cooperation and Development (OECD).


METHODOLOGY
The first step towards collecting current science-based evidence of PMNP effects on human health was a comprehensive literature search across the Web of Science Core Collection (WoSCC) database, using the search phrase "(micro OR nano) AND (plast*) AND (tox*) AND ("in vivo" OR human)".The search was further refined by the additional phrases "("mur*" OR "mouse" OR "rat")", to obtain results published on in vivo rodent animal models.The database was accessed on 28 March 2023, and the search identified a total of 343 papers.They were screened, and non-relevant papers excluded to finally get 43 relevant papers reporting qualitative data on PMNP health effects and quantitative data on PMNP uptake, of which 26 were review articles.Due to the scarcity of humanrelated experimental data, we decided to include and analyse data from in vivo and in vitro studies to fully appreciate the scope of health issues that could be associated with plastic materials and PMNPs.Therefore, our analysis includes all data relating to the magnitude of exposure (i.e., number of particles taken up by cells, tissues, or organisms) and PMNP effects on cell or tissue metabolism.
In addition to the literature search in the WoSCC database, we reviewed information in the AOP-Wiki (16) to identify AOPs that may be associated with the health effects reported for PMNPs.This work was based on comparing adverse effects reported in the 43 WoSCC papers (Figure 1) with molecular initiating events (MIEs) or key events (KEs) described in the AOP-Wiki for different AOPs.The value of the AOP framework lies in the fact that AOPs are not specific for particular stressors but can define downstream PMNP effects if proper MIEs or KEs are identified.AOPs can facilitate risk assessment and management by placing mechanistic knowledge in hazard characterisation in a specific regulatory context (17,18).
Moreover, identifying AOPs relevant for PMNPs can further inform the choice of the most suitable assays for assessing specific adverse outcomes (19).As already demonstrated by several IATA OECD case studies describing AOPs for non-genotoxic carcinogens, skin sensitisation, chemical-induced liver steatosis, and neural development (17), implementation of the AOP framework may improve non-animal in vitro testing for future hazard characterisation of PMNPs.

Routes of exposure and estimated human uptake of PMNPs
Data on routes of human exposure (Figure 2) show that inhalation and ingestion are much more relevant for PMNP uptake than the transdermal route (20,21).There are many sources of inhalation exposure, depending on the type of contamination of indoor and outdoor air.Inhaled PMNPs can translocate into the lung tissue or enter the digestive system via mucociliary clearance (7,22,23).Oral uptake is mostly owed to contamination of bottled drinks or different food items including table salt (24,25).PMNPs can also contaminate food during food processing and packaging.
For all exposure routes, the mechanism of uptake depends on PMNP size, shape, solubility, and surface characteristics as well as on biological factors, such as the site of particle deposition (7,26).Depending on the size, PMNPs can spread through passive diffusion, paracellular transfer, or active cellular uptake (7), which also depends on the cell type, e.g., Peyer's patches in the intestine or alveolar macrophages in the lung (7,27).Even though dermal contact accounts for a relatively small fraction of PMNP uptake, some studies suggest that PMNPs can be absorbed through hair follicles and sweat ducts, which could lead to systemic exposure (28)(29)(30).
Estimations of PMNP intake by humans significantly vary between studies (Table 1) and depend on the type of PMNP and exposure route(s) considered by a particular study.For example, individual dietary PMNP intake in the USA is estimated to 39,000-52,000 particles per year, while the total intake increases to 74,000-121,000 particles per year if we include the inhalation route (31).It is important to note that all estimations presented in Table 1 are based on limited data and may not be representative of a specific population or geographic region.Furthermore, reliable, accurate, and precise methods for identification, characterisation, and quantification of PMNPs in complex matrices like environmental and biological samples are still scarce (8,14,15,20,32).This problem is especially frustrating for the development and implementation of particle dosimetry in PMNP risk assessment and management whose aim is to quantify the number of plastic particles in a particular analytical sample.
Due to the resistance of plastic materials to degradation, bioaccumulation should be considered a serious health issue, as inhaled and ingested plastic particles may remain in different tissues for long and cause chronic health effects (28)(29)(30)33).Deng et al. (34) reported that plastic microparticles in the liver, kidney, and gut tissue accumulate in a range of 0.07-0.41mg of plastic material per gram of tissue.Another animal study (23) reported very low accumulation in the intestinal cell layer and no plastic materials in the liver, spleen, or kidney.Unfortunately, most studies report difficulties in properly assessing the toxicokinetic/toxicodynamic profiles of PMNPs (35).

Health effects of PMNPs
According to the reviewed scientific literature (Figure 1), PMNPs may cause various detrimental effects on cell viability, inflammatory response, lipid metabolism, oxidative status, intestinal microbiota, ion transport, signalling pathways, DNA integrity, hepatic function, and more.Table 2 lists the reported PMNP effects on human and animal cells and tissues.The sheer number of different adverse health effects following exposure to PMNPs raises concern about the ever-increasing plastic pollution.

Linking PMNP-induced health effects to existing AOPs for chemicals
The AOP framework (13) enables collection and logical organisation of experimental data from different sources for efficient identification of essential biological events affected by exposure of living organisms to chemical stressors.Here we used the AOP framework to identify molecular initiating events (MIEs) and key events (KEs) associated with biological effects of PMNPs given in Table 2.The main aim was to generate hypothesis-based AOPs relevant to PMNPs by linking PMNP-induced toxicity endpoints reported so far with existing AOPs in the AOP-Wiki (16).However, it is important to note that many of these AOPs are under development.
Our analysis shows that PMNPs reported to induce biological effects have already been described either as MIEs or as KEs in 170 different AOPs (Table 3), which is an alarming finding that urges for more focused risk assessment of plastic across all steps in its value chain.Table 1 Overview of human-relevant exposure pathways for plastic micro-and nanoparticles with numerical data for levels of intake where available

Ecotoxicity, human cells, humans
Ingestion route: 11,000 particles from shellfish, 4000 particles from drinking water, and 7-1000 particles from edible sea salt per person per year (36) Ecotoxicity, human cells, humans Ingestion route.Sources: seafood, tea bags, honey, sugar, beverage drinks, commercial salts, milk, beer, tap and bottled drinking water (37) Ecotoxicity, humans Ingestion sources: drinking water, food containing plastic particles or weathering from plastic containers, salts and honey, and beer Ecotoxicity, human cells and exposure Ingestion through the food chain (27,31,(38)(39)(40)(41)(42)(43)(44)(45) Human exposure Ingestion route: ≤30 particles/day from tap water and beverages, 37-100 particles/year from sea salt; in total ≤250 pg/kg body weight per day for an adult from tap water, beverages, and sea salt Rodent model 5-day oral exposure to 60 nm polystyrene particles: 10 % of the dose found in the gastrointestinal tract (46) In vitro models Exposure experiments with red blood cells, peripheral blood mononuclear cells, and mast cells (47) In vitro models Exposure experiments with intestinal epithelial cell lines, LS174T, HT-29, and CaCo-2 (22,48) In silico models / (49) Invertebrates and vertebrates Exposure through the food chain (50) In vitro models Exposure experiments with epithelial HeLa and cerebral T98G cells (51) In vivo model organisms Ingestion route: 12,000-204,000 particles per person per year via plastic-contaminated seafood (fish and shellfish), beer, table salt, sugar, and honey In vitro model of the whole digestive system Annual ingestion: 123,000 particles for adults (714 mg/day), 81,000 particles for children (449 mg/day).( 53) In vivo animal models, mammals The primary route of exposure: ingestion of food and water contaminated with PMNP; annual consumption of 39,000 and 52,000 particles per person in the US (57) In vivo Ingestion of PMNP does not provide a significant contribution to the transfer of absorbed chemicals from the water to the biota via the gut (10)

In vitro, in vivo
The oral bioavailability of 50 nm-sized polystyrene particles differs between 0.2 and 2 % in rodents (in vivo) and humans (in vitro); a relationship between the particle's composition, size, and uptake has not yet been established (58) In vivo ecotoxicity (fish)   (63,64), activation of antioxidant enzymes (50), increase in glutathione S-transferase (GST) activity ( 65) and mitogen-activated proteins kinase signalling pathways (66), a decline in lipid digestion and inhibition of digestive enzymatic activities (67), impact on the cell morphology and cell proliferation of immune cells ( Ecotoxicity, animal models, human exposure PS-and PE-MPs; PS-NPs Decrease in hepatic triglyceride and total cholesterol levels, decrease in gene expression related to lipogenesis and triglyceride synthesis in liver, reduction of intestinal mucus secretion (76), metabolic disorders due to alteration of intestinal microbiota (77), induction of IL-6 and IL-8 expression in gastric adenocarcinoma cells (68), induction of oxidative stress inT98G cells (63) Invertebrates and vertebrates

PS-, PE-, HDPE-MPs
Decreased mucus secretion and mucus secretion-related gene expression (79), down-regulation of genes related to ion transport (76), modified serum levels of IL1α and granulocyte colony-stimulating factor G-CSF, decreased regulatory T cell count and increased the proportion of Th17 cells in splenocytes (80), blood neutrophil counts and IgA levels elevated in dams, and spleen lymphocytes were altered in both dams and offspring (81) (46) In vitro [red blood cells, peripheral blood mononuclear cells (PBMCs), mast cells]

PS-MPs
No cytotoxic effects on PBMCs and mast cells, haemolysis of erythrocytes, increased IL-6 production (47) In

In vitro, in vivo PS-MPs
In vitro: no effects on the phosphorylation of STAT-1 and STAT-6, no effect on the expression of CXCL10 and CCL22, CD209 and CD206 genes.
Mice -no statistically significant effects on body and organ weights, no effect on tissue morphology (23) In

CONCLUSIONS
The reviewed literature clearly evidences adverse health effects of PMNPs, even though they are not fully understood in humans.Many studies demonstrate that PMNPs can accumulate and cause inflammation in various tissues and organs or disrupt cellular processes.
It is important to consider all routes to determine aggregate exposure and include all routes in future studies of PMNP health effects.Risk assessment should also include interactions between PMNPs and other environmental pollutants that may act as a Trojan horse introducing hazardous substances into the body.
Research and action are needed to address all these issues and minimise the risks associated with the ever-increasing use of plastic materials.Further studies are needed to fully understand the mechanisms by which PMNPs affect human health and the extent of their toxicity at various exposure levels.Long-term studies are also needed to determine their chronic effects in order to develop effective risk assessment and management strategies and inform policy decisions aimed at minimising exposure to these particles and protecting human health.

Conflicts of interest
None to declare.

Figure 1
Figure 1 Flow chart of literature search in the Web of Science Core Collection (WoSCC) database

Table 2
Science-based evidence of adverse health effects of plastic micro and nanoparticles (PMNP)

Table 3
AOPs in AOP-Wiki related to the observed PMNP effects found in the literature search (see Figure1)[Reported biological effects of PMNP are denoted as molecular initiating events (MIE) or key events (KE) for each AOP.AOPs marked in the last column with an asterisk are "under development"]